Purine derivatives having, in particular, antiproliferative properties, and their biological uses

ABSTRACT

This invention provides 2-, 6, and 9-substituted purine derivatives having, in particular, antiproliferative properties, and suitable for use as pharmaceutical compositions and herbicidal compositions. Also provided are pharmaceutical compositions and herbicidal compositions comprising the 2-, 6, and 9-substituted purine derivatives, and methods of treatment using the 2-, 6, and 9-substituted purine derivatives.

This is a national stage application of PCT/FR96/01905, filed on Nov.29, 1996, which claims priority of French application 95/14237, filedDec. 1, 1995.

The invention relates to new purine derivatives havinganti-proliferative properties and to their biological uses.

It relates in particular to purine derivatives having an inhibitingeffect with respect to cyclin-dependent kinase proteins, or cdk forshort.

The study of the molecular mechanisms which control the cell cycle hasdemonstrated the regulatory role of cdk. These proteins are made up ofat least two sub-units, a catalytic sub-unit (of which cdc2 is theprototype) and a regulatory sub-unit (cyclin). Eight cdk have beendescribed, cdk1 (=cdc2), cdk2-cdk8.

With the exception of cdk3, for which no associated cyclin is known, thecdk are regulated by transitory combination with a member of the cyclinfamily: cyclin A (cdc2, cdk2), cyclin B1-B3 (cdc2), cyclin C (ckd8),cyclin D1-D3 (cdk2-cdk4-cdk5-cdk6), cyclin E (ckd2), cyclin H (cdk7).

Each of these complexes is involved in a phase of the cell cycle. Theactivity of the cdk is regulated by post-translational modification, bytransitory combinations with other proteins and by modification of theirintracellular location. Regulators of the cdk include activators(cyclins, cdk7/cyclin H, cdc25 phosphatases), the sub-units p9^(CKS) andp15^(cdk-BP) and the inhibitory proteins (p16^(NK4A), p15^(INK4B),p21^(Cip1), p18, p27^(Kip1)).

In parallel with purely fundamental research into the regulatorymechanisms of cell division, the importance of dysregulations ofcyclin-dependent kinases in the development of human tumours has beendemonstrated by several studies. Over-expression of cyclins D and E inseveral tumours, over-expression of cdc2, the oncogenic properties ofcyclins D and A, abnormal temporary expression of cyclin-dependentkinases and major dysregulation of protein inhibitors (mutations,deletions) have thus been found.

The regulators of the cell division cycle are the subject of a largenumber of clinical studies (use as indicating markers for treatment).

These results greatly encourage efforts for detailed comprehension ofthe regulatory mechanisms of the cell cycle. They also lead to thesearch, by screening for molecules which inhibit cyclin-dependentkinases.

Several kinase inhibitors have been described, such as butyrolactone,flavopiridol and 2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine,called olomoucine. Works relating to olomoucine are reported by Veselyet al. in the article carrying the reference (1) in the list ofbibliographic references given at the end of the description.

This cdc2 inhibitor of high efficacy (its IC₅₀ is 7 μM) and highselectivity (more than 35 kinases have been tested) corresponds to theformula:

The works of the inventors in this field have led them to develop newmolecules of particular interest which inhibit cdc2 in low doses, whilemaintaining the enzymatic specificity of olomoucine.

The object of the invention is therefore to provide new purinederivatives having, in particular, anti-proliferative properties.

The invention also relates to a process for obtaining these derivativesby synthesis, which enables them to be prepared on an industrial scale.

It also relates to their therapeutic use and their use as a herbicide.

The purine derivatives of the invention are characterized in that theycorrespond to the formula I.

in which

R2, R6 and R9, which are identical to or different from one another,represent a halogen atom or an R-NH-, R-NH-NH-, NH₂-R′-NH- orR-NH-R′-NH- radical, in which R represents a straight- orbranched-chain, saturated or unsaturated alkyl radical, an aryl orcycloalkyl radical or a heterocyclic ring and R′ represents a straight-or branched-chain, saturated or unsaturated alkylene group or an aryleneor cycloalkylene group, R and R′ each containing 1 to 8 carbon atoms andbeing substituted, where appropriate, by one or more -OH, halogen, aminoor alkyl groups.

R2 can also represent a heterocyclic ring carrying, where appropriate, astraight- or branched-chain, saturated or unsaturated alkyl radical, anaryl or cycloaryl radical or a heterocyclic ring, optionally substitutedby one or more -OH, halogen, amino or alkyl groups.

R9 can also represent a straight- or branched-chain, saturated orunsaturated alkyl radical or an aryl or cycloalkyl radical,

R2 and R9 can also represent a hydrogen atom, with the exception of thederivatives in which the said substituents have, respectively, thefollowing meanings:

R6 and R9 - a benzylamino and methyl group,

R2 and R6 - a hydroxyethylamino and benzylamino group,

R2, R6 and R9 - an amino, benzylamino and methyl, or chloro, amino andmethyl, or chloro, benzylamino and methyl, or chloro,3-hydroxybenzylamino and methyl, or chloro, 5-hydroxypentylamino andmethyl, or 2-hydroxyethylamino, benzylamino and isopropyl, or2-hydroxyethylamino, amino and methyl, or 2-hydroxyethylamino,isopentenyl and methyl, or 2-hydroxyethylamino, isopentenylamino andmethyl, or 2-hydroxyethylamino, benzylamino and methyl, or2-hydroxyethylamino, benzylamino and 2-hydroxyethyl, or2-hydroxyethylamino, benzylamino and isopropyl, or 2-hydroxyethylamino,(3-hydroxybenzyl)amino and methyl, or 2-hydroxyethylamino,(3-hydroxybenzyl)amino and isopropyl, or 2-hydroxyisobutylamino,6-benzylamino and methyl, or 2-hydroxyethylamino, isopentenylamino andisopropyl, or (2-hydroxyethyl)amino, (4-methoxybenzyl)amino andisopropylamino group.

and the purine derivatives of the invention are furthermorecharacterized in that they have an IC₅₀ less than or equal to about 5 μMfor cdc2/cyclin B.

The abovementioned derivatives which are excluded from the invention aredescribed in reference (1).

In general, the derivatives of the invention are kinase proteininhibitors of great interest.

Preferably, the halogen atom is chosen from chlorine, bromine orfluorine, the alkyl radical is chosen from the methyl, ethyl, propyl,isopropyl, butyl and isobutyl, pentyl, hexyl and heptyl radicals thealkylene radical is chosen from the methylene, ethylene, propylene,isopropylene, butylene, isobutylene, pentene or isopentene radicals, thearyl radical is a benzyl group, the cycloalkyl radical is a cyclohexylgroup, the arylene radical is a benzylene group, the cycloalkyleneradical is a cyclohexylene group and the heterocyclic ring is anitrogen-containing and or oxygen-containing heterocyclic ring, such asan imidazole, an oxadiazole, a pyridine, a pyridazine or a pyrimidine,or also a pyrrolidine.

According to one embodiment of the invention, R2 is chosen from theradicals which are capable of bonding in a cdk2/ATP complex to a regionof the bonding domain of ATP occupied by ribose. These areadvantageously radicals chosen from a chlorine atom, and amino,methylamino, ethylamino, n-heptylamino, aminoethylamino,aminopropylamino, dimethyleminoethylamino, hydroxyethylamino,hydroxy-propylamino, hydroxyisobutylamino, hydroxypentylamino,dimethylhydrazino or hydroxymethylpropylamino.[(2R)-2-hydroxymethyl-pyrrolidin-1yl], N-benzyl-aminoethanol,(R,S)-amino-hexanol, (S)-amino-2-phenylethanol,(R)-amino-2-phenylethanol, (R)-amino-3-phenylpropanol,(R,S)-amino-pentanol, (R)-amino-propanol, (S)-amino-propanol and(R)-N-pyrrolidine-methanol radical.

Particularly preferred radicals contain a hydroxypropylamino radical asthe group R2.

According to another embodiment of the invention, R6 is chosen from anamino, isopentenylamino, hydroxypentylamino,4-hydroxy-3-methyl-trans-2-butenylamino, benzylamino,hydroxybenzylamino, hydroxyethylbenzylamino, cyclohexylmethylamino,isopentene, benzylamino or (3-iodo)-benzylamino group.

R6 preferably comprises a hydrophobic radical, such as benzyl,hydroxybenzyl or isopentenyl.

Preferably, R2 is chosen from the group consisting of[1-D,L-hydroxymethylpropylamino], [(2R)-2-hydroxymethyl-pyrrolidin-1-yl]and [(R)-N-pyrrolidine-methanol] and R6 is benzylamino.

According to yet another embodiment of the invention, the substituent R9is chosen from a hydrogen atom and a methyl, isopropyl or hydroxyethylradical.

R9 is advantageously a hydrophobic group, in particular the isopropylgroup.

Preferred purine derivatives of the invention are chosen from thecompounds in which R2, R6 and R9 are as indicated in the following table1:

TABLE 1 IC₅₀ μM cdc2/cyclin R2 R6 R9 B 3-hydroxypropylamino benzylaminoisopropyl 1 2-hydroxypropylamino benzylamino isopropyl 0.9t-D,L-hydroxymethyl- benzylamino isopropyl 0.65 propylaminoaminoehtylamino benzylamino isopropyl 1 2-hydroxypropylamino isopentenylisopropyl 1.2 2-hydroxypropylamino cyclohexyl- methyl 4 methylaminochloro isopentenyl- isopropyl 2.5 amino (2R)-2-hydroxymethyl-benzylamino isopropyl-(9H) 0.45 pyrrolidin-l-yl N-benzylaminoethanolbenzylamino isopropyl-(9H) 2.5 (R,S)-amino-hexanol benzylaminoisopropyl-(9H) 2.5 (S)-amino-2- benzylamino isopropyl-(9H) 4.3phenylethanol (R)-amino-2- benzylamino isopropyl-(9H) 1 phenylethanol(R)-amino-3- benzylamino isopropyl-(9H) 2.7 phenylethanol(R,S)-amino-pentanol benzylamino isopropyl-(9H) 0.9 (R)-amino-propanolbenzylamino isopropyl-(9H) 0.85 (S)-amino-propanol benzylaminoisopropyl-(9H) 1 (R)-N-pyrrolidine- (3-iodo)- isopropyl-(9H) 0.45 μMmethanol benzylamino (R)-N-pyrrolidine- benzylamino cyclopentyl- 0.7methanol (9H)

The following derivatives are particularly preferred, that is to say:2-1-D,L-hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine,non-crystalline6-benzylamino-2-[(2R)-2-hydroxymethyl-pyrrolidin-1-yl]-9-isopropyl-(9H)-purine,2-(R)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-2-phenylethanol,2-(R,S)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-pentanol,2-(R)-[6-benzylamino-9-isopropyl-(9H)-purin-2--yl]-amino-propanol,2-(S)-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-propanol,2-(R)-(-)-[6-(3-iodo)-benzylamino-9-isopropyl-(9H)-purin-2-yl]-N-pyrrolidine-methanoland2-(R)-(-)-[6-benzylamino-9-cyclopentyl-(9H)-purin-2-yl]-N-pyrrolidine-methanol.

The invention also relates to the optical isomers and the racemicmixtures and, where appropriate, the geometric isomers of thederivatives defined above, in particular the R isomer of(2-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-2-phenylethanol andof 2-[6-benzylamino-9-isopropyl-(9H)-purin-2-yl]-amino-propanol.

The derivatives defined above are obtained by the conventional methodsof organic synthesis. A starting purine derivative of which thesubstitutions allow introduction of the desired groups is used.

For example, using a 2-chloro-6-benzylamino derivative of purine, it ispossible to introduce an alkyl group in position 9 by reaction with, forexample, the corresponding alkyl halide.

Reaction with an aminoalcohol then allows introduction of analkylhydroxyalkylamino group in position 2, in place of the chlorogroup.

According to an aspect of great interest, the derivatives of theinvention have inhibitory properties on kinases of high selectivity.These inhibitory effects are reversible.

The cdk play a central role in the initiation, development andachievement of the events of the cell cycle, and the inhibitorymolecules of cdk are capable of limiting undesirable cell proliferation,such as cancer, psoriasis and growth of fungi and parasites (animals,protists), and also of plants (herbicides), and of intervening in theregulation of neurodegenerative diseases, such as neuronal apoptosis andAlzheimer's disease.

The kinases which are more specifically sensitive to the inhibitoryeffects of these derivative are the cdc2, the cdk2 and cdk5.

Their inhibition is obtained with very low doses of purine derivatives.

An IC₅₀ with respect of cdc2 of less than 50 μM, and even than that ofolomoucine (7 μM), which is regarded, however, as a potent inhibitor,has thus been observed most generally.

The invention particularly relates to purine derivatives having an IC₅₀which does not exceed 5 μM, and especially2-(1-D-L-hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine, alsocalled roscovitine below, the IC₅₀ of which is 0.65 μM, non-crystalline6-benzylamino-2-[(2R)-2-hydroxymethyl-pyrrolidin-1-yl]-9-isopropyl-(9H)-purineand2-(R)-(-)-[6-(3-iodo)-benzylamino-9-isopropyl-(9H)-purin-2-yl]-N-pyrrolidine-methanol.

This derivative, which is an inhibitor of high efficacy and highselectivity with respect to the cdk, cdc2, cdk2 and cdk5, unexpectedlyhas in return effects on the kinases erk 1 and erk 2 similar to those ofolomoucine. The selectivity is thus clearly superior with respect tocyclin-dependent kinases. This advantage, which is found with the otherpurine derivatives of the invention, allows elimination of interferenceswith the transduction routes of signals further upstream which involvethe kinases erk 1 and erk 2 in several cell responses other than celldivision.

The invention also relates to complexes of purine derivatives with thecdk, and especially to the crystallized form of the complex of cdk2 androscovitine.

Studies carried out on the derivatives of the invention have shown, inaddition to their specific inhibitory properties on kinases, celleffects and effects on apoptosis of great interest.

At a very low concentration (micromolar for roscovitine and a largenumber of derivatives), they are thus capable of inhibitingprophase/metaphase transition, as shown by experiments carried out onovocytes of starfish and sea urchin embryos, which are reported in theexamples.

On acellular Xenopus extracts, they are capable of inhibiting both thepromoter factor of the M phase and DNA synthesis.

These cell effects are advantageously obtained at very lowconcentrations of derivatives.

It is known that various works relate to the relationships which existbetween the cell cycle and apoptosis. Various routes lead to apoptosisof cells, some of which are dependent on kinases and others of which, incontrast, do not seem to require these enzymes. It has been demonstratedthat apoptosis can be induced at the G1 and G2 stage, and that followingdamage to the DNA, some cells stop at the G1 stage and an apoptoticroute dependent on p53 is thus induced.

In other situations, it seems that the cells stop at the G2/M stage, inresponse to damage caused to the DNA, and activation of an apoptoticp53-independent route is observed.

This route proves to be particularly important for treatment of tumoursin which a loss of active p53 is found.

The benefit of having available, with the derivatives of the invention,means for stimulating a p53-independent apoptosis in cells which havestopped at the G2 stage by damage to the DNA with the aid of agents suchas mitoxantrone or cis-platin is thus estimated.

The cdc2 inhibitors of the invention can thus increase the therapeuticeffects of the anti-tumoral agents currently used.

As cdk5 inhibitors, the derivatives of the invention can also play arole in reducing abnormal hyperphosphorylation of tau observed duringAlzheimer's disease.

To these various advantageous properties is added the benefit of absenceof cytotoxicity of the derivatives of the invention.

The invention thus relates to the utilization of the properties of thesederivatives, in particular their antimitotic and antineurodegenerativeproperties, for formulation of pharmaceutical compositions.

The pharmaceutical compositions of the invention are characterized inthat they comprise an effective amount of at least one purine derivativeas described above, in combination with an inert pharmaceutical vehicle.

The compositions of the invention are particularly suitable asantimitotic medicaments, in particular for chemotherapy of cancers, oralso for treatment of psoriasis, parasitoses, such as those caused byprotists or fungi, or Alzheimer's disease, or neuronal apoptosis.

These compositions comprise, where appropriate, active principles ofother medicaments. There may be mentioned, in particular, theircombination with antimitotic medicaments, such as those based on taxol,cis-platin, agents for intercalation of DNA and others.

The conditioning with respect to sale, in particular labelling andinstructions for use, and advantageously packaging, are formulated as afunction of the intended therapeutic use

The pharmaceutical compositions of the invention can be administered invarious forms, more specifically by an oral or injectable route.

For administration by the oral route, compressed tablets, pills,tablets, capsules and drops are used in particular. These compositionsadvantageously comprise 1 to 100 mg of active principle per dose unit,preferably 10 to 40 mg.

Other forms of administration include injectable solutions for theintravenous, subcutaneous or intramuscular route, formulated fromsterile or sterilizable solutions. They can also be suspensions oremulsions.

These injectable forms comprise 1 to 50 mg of active principle,preferably 10 to 30 mg, per dose unit.

By way of indication, the dosage which can be used in man corresponds tothe following doses for example, 10 to 50 mg/day are thus administeredto the patient in one or more doses for treatment of tumours or to treatpsoriasis parasitoses.

The invention also relates to herbicidal compositions comprising atleast one purine derivative as defined above, optionally in combinationwith other phytopharmaceutical agents.

The invention also relates to biological reagents, the active principlesof which consist of the purine derivatives defined above.

These reagents can be used as references or standards in studies of celldivision.

Other characteristics and advantages of the invention are described inthe examples which follow with reference to FIGS. 1 to 8, in which

FIG. 1 shows the results of kinetics under linear conditions from testsrelating to the activity of p34^(edc2)/cyclin B at variousconcentrations of roscovitine,

FIG. 2 shows the percentage breakdown of the germinal vesicle ofovocytes of the starfish as a function of the concentration ofroscovitine,

FIGS. 3 and 4 show respectively, the effects of roscovitine onmaturation of ovocytes of the starfish and dephosphorylation ofp34^(cdc2) tyrosine in vivo,

FIG. 4 shows the effects of roscovitine on the mitotic cycle of seaurchin embryos,

FIG. 5 shows these embryos stopped at the late prophase stage and

FIG. 6 shows the effects of roscovitine on the synthesis of DNA in vitroand the MPF activity,

FIG. 7 shows the effects of roscovitine on the inhibition of the growthof L1210 cells and the halting of their cell cycle at G2/M, in FIG. 7Athe growth of L1210 cells after exposure to various concentrations ofroscovitine (mean ± standard deviation in relation to the growth of theuntreated control cell) is shown, and in FIG. 7B the means (± standarddeviation) of the distribution over the cycle of cells which have firstbeen cultured for 48 hours in the presence or absence of 60 μMroscovitine are shown,

FIG. 8 shows the inhibitory effect of roscovitine on in vivophosphorylation of vimentine at sites specific to cdc2.

MATERIAL AND METHODS Chemical products

Sodium orthovanadate, 1-methyladenine (1MeAde), EGTA, EDTA, MOPS,β-glycerophosphate, dithiothreitol (DTT), sodium fluoride, nitrophenylphosphate, leupeptin, aprotinia, soya trypsin inhibitor, benzamidine,histone H1(type III-S), basic myelin protein, casein, protaminesulphate, isopropyl β-D-thiogalactopyranoside (IPTG), Sepharose 4Bactivated with CNBr, LB medium, glutethione and glutathione-Sepharosebeads; all these products are such as those marketed by Sigma Chemicals.

The purine analogues are generally dissolved such that stock solutionsof 100 mM in DMSO are available. The final concentration in DMSO in thereaction mixture is less than 1% (v/v).

[y-³²P]-ATP is a product of Amersham.

The GST-retinoblastoma protein is expressed in bacteria and purifiedover glutathione-Sepharose beads as described previously in (1) and (2).

Buffers

Fiomogenization buffer:

60 mM β-glycerophosphate, 15 mM p-nitrophenyl phosphate, 25 mM MOPS (pH7.2), 15 mM EGTA, 15 mM MgCl₂, 1 mM DTT, 1 mM sodium vanadate, 1 mM NaF,1 mM phenyl phosphate, 10 μg leupeptin/ml, 10 μg aprotinin/ml, 10 μgsoya trypsin inhibitor/ml and 100 μM benzadmidine.

Buffer C:

Composition of the homogenization buffer but with 5 mM EGTA, without NaFand without protease inhibitors.

Preparation of extracts of starfish ovocytes in phase M

To obtain preparations of ovoctye extracts on a large scale, the gonadsof mature Marthasterias glacialis are removed and are incubated with 10μM 1-MeAde in natural sea-water filtered over Millipore, until the eggsare laid. The ovecytes have thus all entered phase M. They are separatedoff from the incubation medium by centrifugation, frozen directly inliquid nitrogen and kept at −80° C. (see (1) and (3)).

The ovocytes in phase M are homogenized in the homogenization buffer inan amount of 2ml/g ovocytes.

After centrifugation for 45 minutes at 100,000 g, the supernatant isrecovered and used directly for purification of the p34^(cdc2)/cyclin Bkinase by affinity chromatography over p9^(CKShs1) Sepharose beads (see(1) and 4)).

Enzymes

The activities of the kinases are determined at 30° C. in buffer C bymeans of counter-indication. The blank values are subtracted from thedata and the activities are calculated in pmol of phosphate incorporatedin the protein acceptor for an incubation of 10 minutes.

The controls are used with suitable dilutions in DMSO.

The phosphorylation of the substrate is determined, where appropriate,by autoradiography after SDS-PAGE.

p34^(cdc2)/cyclin B is purified from the phase M ovocytes of thestarfish by affinity chromatography over p9^(CKSml)-Sepharose, wherethey are eluted with the aid of p9^(CKSml) as described above (see (2),(3), and 5)).

For the determination, 1 mg histone H1 (Sigma type III-SV)/ml in thepresence of 15 μM [y-³²P]-ATP (3,000 Ci/mmol, 1 mCi/ml) in a finalvolume of 30 μl is used (see (1) and (6)).

After an incubation time of 10 minutes at 30° C., aliquots of 25 μl ofsupernatant are deposited on Whatman P81 phosphocellulose paper and,after 20 seconds, the filters are washed 5 times (for at least 5 minuteseach time) in a solution of 10 ml phosphoric acid per liter of water.

The damp filters are transferred to 6 ml plastic scintillation ampoules,5 ml SCS scintillation liquid (Amersham) are then added and theradioactivity is measured in a Packard counter.

The kinase activity is expressed in pmol of phosphate incorporated intohistone H1 for an incubation of 10 minutes or in per cent of the maximumactivity.

To carry out the kinetic experiments under linear conditions, the testsystem up to the final point for the p34^(cdz2) kinase is used, asdescribed, but, on the basis of preliminary tests, suitable unsaturatedconcentrations of substrate are used.

The p34^(cda2)/cyclin B kinase is added to obtain a linear activity withrespect to the concentration of enzyme and to time.

In the majority of cases, this requires a 3- to 10-fold enzymaticdilution in buffer C.

The rate data are expressed in pmol incorporated into the substrate persecond per amount of enzyme added. The apparent inhibition constants aredetermined by analysis by graph.

p33^(cdc2)/cyclin A and p33^(cdk2)/cyclin E are reconstituted fromextracts of sf9 insect cells infected with various baculoviruses.

Cyclins A and B are fusion proteins of GST-cyclins and the complexes arepurified over glutathione-Sepharose beads.

The kinase activities are determined with 1 mg/ml histone H1 (Sigma,type IIIS) in the presence of 15 μM [y-³²p]-ATP for 10 minutes in afinal volume of 30 μl, as described for the p34^(cdc2)/cyclin B kinase.

p33^(cdk3)/p25 is purified from the bovine brain (7), but the Mono Schromatography stage is not used.

The active fractions recovered from the Superose 12 column are combinedand concentrated to a final concentration of about 25 μg enzyme/ml.

The determination of the kinase is carried out with 1 mg/ml histone H1(Sigma, type IIIS) in the presence of 15 μM [y-³²P]-ATP over 10 minutesin a final volume of 30 μl, as described for p34^(cdc2)/cyclin B.

p33^(cdk4)/cyclin D1 is obtained from lysates of insect cells. cdk4 is aGST-cdk4 construction product and the active complex is purified overglutathione-Sepharose beads.

Its kinase activity is determined with a purified GST-retinoblastomaprotein in the presence of 15 μM [y-³²P]-ATP in a final volume of 30 μl.

After incubation for 15 minutes, Laemmli buffer (2×30 μl) is added.

The phosphorylated substrate is resolved by 10% SDS-PAGE and analysed byautoradiography by exposure to MP Hyperfilm for about 14 hours anddensitometry.

p33^(cdk6)/cyclin D2 is obtained from lysates of insect cells 8). Forthe tests, the procedure is as indicated above for the p33^(cdk4)/cyclinD1 protein.

The MAP kinases: GST-erk1 (9) cloned from a human HepG2 bank isexpressed in bacteria, purified over glutathione-Sepharose beads andtested with 1 mg basic myelin protein per ml in the presence of 15 μM[y-³²P]-ATP as described above for the p34^(cdc2)/cyclin B kinase.

The erk1 and erk2 proteins marked with the aid of histone are activatedin vitro by MAPKK, purified (affinity-Ni and Mono Q) and tested asdescribed above for 10 minutes in a final volume of 20 μl.

The catalytic sub-unit of the cAMP-dependent kinase, purified from thebovine heart, is tested with 1 mg histone H1 per ml in the presence of15 μM [y-³²P]-ATP as described above for p34^(cdc2)/cyclin B.

The cGMP-dependent kinase (10), purified to homogeneity from the smoothmuscle of the trachea of bovine origin, is tested with 1 mg histone H1per ml in the presence of 15 μM [y-³²P]-ATP as described above forp34^(cdc2)/cyclin B.

The casein 2 kinase is isolated from cytosol of the liver of the rat(11) and tested with 1 mg casein per ml and 15 μM [y-³²P]-ATP. Thesubstrate is deposited on Whatman 3MM filters and washed with 10% TCA(w/v).

The short-chain myosin kinase purified from chicken gizzards (12) istested in the presence of 100 nM calmodulin, 100 μM CaCl₂, 50 mM HEPES,5 mM MgCl₂, 1 mM DTT and 0.1 mg BSA/ml at pH 7.5 using a syntheticpeptide on the basis of the phosphorylation site of the light chain ofmyosin of the smooth muscle (KKRPQRATSNVFAM, 50 μM) and in the presenceof 15 μM [y-³²P]-ATP in a final volume of 50 μl.

The incorporation of radioactive phosphate is checked onphosphocellulose filters as described above.

The homologous ASK-y in the GSK-3 plant is expressed as GST fusionprotein in E. coli (13) and purified over glutathione-Sepharose. Theactivity of the ASK-y kinase is determined, over 10 minutes at 30° C.,with 5 μgm basic myelin protein in the presence of 15 μM [y-³²P]-ATP ina final volume of 30 μl. The basic phosphorylated myelin protein isrecovered on Whatman P81 phosphocellulose paper as described above forp34^(cdc2)/cyclin B.

The kinasic tyrosine domain for the insulin receptor (14) isover-expressed in a baculovirus system and purified to homogeneity. Itskinase activity is determined over 10 minutes at 30° C. with 5 μgRaytide (Oncogene Sciences) in the presence of 15 μM [y-³²P]-ATP in afinal volume of 30 μl. The phosphorylated Raytide product is recoveredon Whatman P81 phosphocellulose paper as described above forp34^(cdc2)/cyclin B.

Example 1: Synthesis of roscovitine

The synthesis is carried out in 3 stages and comprises thepreparation 1) first of 6-benzylamino-2-chloropurine, then 2) of6-benzylamino-2-chloro-9-isopropylpurine, and 3) of6-benzylamino-2-R-(1-ethyl-2-hydroxyethylamino)-9-isopropylpurine.

1) Synthesis of 6-benzylamino-2-chloropurine:

The procedure is as described by Hocart in Phytochemistry 1991, 30,2477-2486.

2) Synthesis of 6-benzylamino-2-chloro-9-isopropylpurine (I):

A mixture of 6-benzylamino-2-chloropurine (3.7 g; 14.2 mmol), potassiumcarbonate (11 g; 8 mmol) and isopropyl bromide 8.2 ml; 87 mmol) in 100ml absolute DMSO is stirred at room temperature for three days. Theabsence of 6-benzylamino-2-chloropurine is confirmed by thin layerchromatography [CHCl₃-MeOH (98:2)]. The DMSO and the excess isopropylbromide are removed by distillation in vacuo at below 50° C. The residueis partitioned between water and ethyl acetate. The organic phase isdried over Na₂SO₄ and evaporated in vacuo.

Crystallization in MeOH gives 3.51 g (82%) of product; m.p. 181-182° C.,UV (MeOH): λ_(uax4) 273.5, IR (Nicolet 205, KBr, DRIFT cm 1713, 1626,1572, 1537, 1497, 1471, 1456, 1425, 1398, 1355, 1314, 1292, 1255, 1228,1202.

3) Synthesis of6-benzylamino-2-R-(1-ethyl-2-hydroxyethylamino)-9-isopropylpurine (II),racemic derivative:

A sealed ampoule, in which a vacuum has been established, containing 2.7g (8.95 mmol) I and 17 ml (0.18 mol) R(-)-2-amino-1-butanol (Fluka 90%,R,S>9:1) is heated in an oven at 160-165° C. for 3 h 30 min. The excessamine is evaporated off at a temperature below 50° C. and the produce IIis purified over a chromatography column using increasing amounts ofMeOH in CHCl₃, that is to say 0, then 2, and 3%.

Crystallization in ethyl acetate gives 2.2 g II (69%), m. p. 132-134°C., [α]=÷35.1 (c=0.29, CHCl). Mass spectrometry (Finnigam MAT 90, BEgeometry 70 eV, temperature of the source 250° C., emission current 1mA, acceleration voltage 5 keV, direct entry, DIP temperature between190-220° C.]. HRMS was carried out by the method of overlapping peaksusing Ultramark 1600 F. (PCR Inc., Fla. USA) as the standard] 354.2167(M^(+, C) ₁₉H₂₆N₆O Calc. 354.2168, 27%), 325 (7%), 324 (29%), 232(100%), 295 (3%), 282 (7%), 281 (3%), 217 (6%), 185 (5%) 134 (3%), 91(34%). FTIR (Nicolet 205, KEr, DRIFT. cm⁻¹): 1622, 1610, 1547, 1540,1452, 1389, 1370, 1261, 1068.

Example 2: Study of the inhibitory properties on kinases of roscovitineand its effects on the cell cycle

a) Study of the inhibitory properties on kinases

The enzyme activities shown in the following table were measured afteraddition of roscovitine or olomoucine at increasing concentrations.These activities were measured with suitable substrates (histone H1,basic myelin protein, casein etc. . . ) with 15μM ATP.

The IC₅₀ were calculated from the dose/response curves obtained. Thesymbol (-) indicates that no inhibitory effect was observed. The highestconcentration tested is given in parentheses.

TABLE 2 IC₅₀ (μM) Enzyme Roscovitine Olomoutine cdc2/cyclin B 0.65 7cdc2/cyclin A 0.7 7 cdc2/cyclin E 0.7 7 cdc4/cyclin D1 >1000 >1000cdk5/P35 0.16 3 cdk5/cyclin D3 >500 >250 GST-erk1 30 30 erk1 34 50 erk214 40 cAMP-dependent PK >1000 >2000 cGMP-dependent PK −(1000) >2000Light chain myosin kinase 90 >1000 Casein 2 kinase −(1000) >2000 ASK-γ(GSK-3 plant) 220 130 Insulin receptor tyrosine kinase 70 4000 c-src 250— v-abl >1000 —

Inhibition of cdc2, cdk2 and cdk5.

Examination of these results shows that roscovitine has an activitywhich is 10 times higher than olomoucine with respect to the targetscdc2 and cdk2 and 20 times higher with respect to cdk5.

By comparison, its effect seems limited, as observed with olomoucine, onthe cdk4/cyclin D1 and cdk6/cyclin D2 kinases (the IC₅₀ are greater than100 μM). This absence of an effect was confirmed with cdk4 originatingfrom various sources. Working under identical conditions.GST-p16^(INK4A) inhibits cdk4/cyclin D1.

Specificity of the inhibitory effect

As can be seen, the majority of the kinases are inhibited weakly or notat all.

Although roscovitine has an efficacy at least 10 times greater than thatof olomoucine with respect to its cdk targets, its inhibitory effect isvery similar to that of olomoucine with respect of erk1 and erk2. A40-fold higher concentration of roscovitine thus seems necessary toinhibit erk1 (20-fold for erk2) in a manner similar to the inhibition ofedc2.

b) Effect on ATP

To study the action mechanism of roseovitine, kinetic experiments werecarried out in the presence of increasing concentrations of roscovitinevarying the levels of ATP (from 0.1 to 0.5 mM), the concentration ofhistone H1 being kept constant at 0.7 mg/ml.

The results are shown on FIG. 1.

These results demonstrate that roscovitine acts as a competitiveinhibitor for ATP. Taking account of the linearity of the slopes as afunction of the concentrations of roscovitine, it is called a linearinhibitor. The apparent inhibition constant Ki is 1.2 μM.

Analysis of the structure of the co-crystal of roscovitine and cdk2confirms that, like olomoucine, roscovitine bonds to ATP in the bondingpocket and that its purine ring is orientated in the same way as that ofolomoucine, that is to say in a totally different manner to the purinering of ATP.

c) Study of the effect on the synthesis of DNA and the MPF activity.

The results of experiments carried out on several cell types aredescribed.

Effect on the maturation of ovocytes of the starfish and on thedephosphorylation of p34^(cdc2) tyrosine in vivo.

The ovocytes of starfish, stopped at the prophase, are treated for 15minutes with increasing concentrations of roscovitine before addition ofthe hormone 1-MeAde (1 μM). After 30 minutes, the % breakdown of thegerminal vesicle (GVBD) is recorded. These values are shown on FIG. 2 asa function of the concentration of roscovitine (in μM). Roscovitineinhibits breakdown on the nuclear envelope with an IC₅₀ of 5 μM (theIC₅₀ of olomoucine working under the same conditions, is 30 μM). Theseresults are given on FIG. 2.

As already observed with olomoucine, roscovitine reduces, but does notinhibit, the dephosphorylation of p34^(cdc2) tyrosine in vivo. Theovocytes are treated with 10 μM roscovitine for 15 minutes beforeaddition of 1 μM 1-MeAde at time 0. The extracts are prepared at varioustimes and introduced on to a column of p9cksns1-Sepharose beads.

The proteins bonded to the beads are resolved by SDS-PAGE beforecarrying out a western blot with anti-PSTAIRE antibodies. A photographof the western blot is shown on FIG. 3. The phosphorylated forms ofp34^(cdc2) appear in the upper part and the dephosphorylated formsappear in the lower part.

Roscovitine therefore inhibits not the activation of cdc2 but itsactivity. The dephosphorylation of p34^(cdc2) tyrosine is catalysed bycdc25 and normally precedes the activation of the cdc2 kinase at theG2/M transition. Furthermore, the cdc2 kinase phosphorylates andover-activates cdc25 phosphatase. Roscovitine has thus been able tocause interruption at the cdc2 kinase level, bringing about a reductionin the dephosphorylation.

Effects on the mitotic cycle of sea urchin embryos.

Roscovitine is added 60 minutes after fertilization. The percentage ofembryos which have divided is recorded 120 minutes after fertilization.The results are given on FIG. 4.

It is found that is causes a dose-dependent halt at the late prophasestage.

The IC₅₀ is 10 μM. (Even at 100 μM, olomoucine causes only a slowingdown of the prephase/metaphase transition, but does not stop the cellsat the prophase).

A large nucleus is observed in the eggs stopped in this way byroscovitine, as shown on FIG. 5.

This halt proves to be totally reversible. In fact, after severalwashing with sea-water, the eggs enter the mitotic cycles again anddevelop into normal pluteus larvae. These results are obtained even atelevated concentrations of roscovitine of 100 μM.

Effects on the synthesis of DNA in vitro and the MPF activity inextracts of Xenopus eggs.

The tests are carried out in accordance with (15), working as describedin (1) for olomoucine.

The extracts of Xenopus stopped at the metaphase stage are incubatedwith roscovitine and sperm chromatin.

With concentrations of roscovitine ranging from 0 to 5 μM, thechromosomes remain highly condensed and no nuclear envelope is visible.At a concentration of 10 μM and above, interphase nuclei appear, withthe chromatin partly decondensed and an intact nuclear envelope, showingthat the MPF activity has been inhibited (the IC₅₀ is 5 μM).

The inhibition of DNA synthesis has also been studied, proceeding asdescribed in (1) for olomoucine.

Roscovitine and sperm chromatin were thus added to an extract of eggswhich had been stopped at the metaphase stage.

The extract was then abandoned at the interphase stage by addition ofCaCl₂(15) and (16). The synthesis of total DNA was measured 3 h later byincorporation of [y-³²P]-dATP into material which can be precipitatedwith TCA.

As shown in FIG. 6, the replication is inhibited by roscovitine with anIC₅₀ of 15 μM.

The invention thus provides new purines having inhibitory properties oncdc2/cyclin B of high specificity.

Example 3 Biochemical properties and effects of roscovitine on mammaliancells. Method

In vitro screening of human tumoral cells

Sixty cell lines of human tumours comprising nine types of tumour werecultured for 24 hours prior to continuous exposure for 48 hours to0.01-100 μM roscovitine. To estimate the cytotoxicity a sulphorhodamineB protein test was used.

Culture of the L1210 cell

L1210 cells sampled from cultures in exponential growth on RPMI-1640medium supplemented with 10% foetal calf serum, penicillin andstreptomycin were counted with the aid of a haemocytometer, placed in anamount of 5×10⁴ cells per milliliter in 96-well tissue culture plates inthe presence or in the absence of various concentrations of roscovitineor olomoucine, and then incubated at 37° C. under 5% CO₂. To reverse theeffect of roscovitine, L1210 cells cultured for two days in the presenceor absence of roscovitine were washed in PBS to remove any trace ofactive product, counted and placed again in fresh medium containing noactive product (roscovitine or olomoucine). The cell growth was measureddaily using the tetrazolium microculture test. The analysis of the cellcycle was carried out on cells fixed in ethanol, treated with 100 μg/mlRNase and stained with propidium iodide. Data acquisition was achievedwith the aid of a Coulter flow cytometer (Hialeah, Fla., USA) EPICSElite (registered trademark), and these data were analysed with the aidof Multicycle software (Phoenix Flow Systems, San Diego, Calif. USA)(registered trademark). All the tests were carried out with threerepeats and all the experiments were repeated at least twice.

In vivo phosphorylation of vimentine

To study the in vivo phosphorylation of vimentine by the cdc2 kinase,the cells were either not treated or treated with 60 μM roscovitine for48 hours prior to exposure to 10 ng/ml colcemide for an additional 2hours. The cell extracts were then placed on a 10% SDS-PAGE gel formigration, transferred by western blots and incubated with 4A4antibodies. These antibodies undergo a cross-reaction with vimentinephosphorylated by cdc2, but react neither with vimentine phosphorylatedby other kinases (cAMP-dependent kinase protein, C kinase protein,Ca²⁺-calmodulin-dependent kinase protein), nor with non-phosphorylatedvimentine; the 4A4 antibodies specifically recognize vimentine which isphosphorylated at its Ser-55 residue by the cdc2 kinase when the cellenters mitosis.

Results

Roscovitine (0.01-100 μM; exposure for 48 hours) was tested on 60 humantumoral cell lines comprising nine types of tumours (leukaemia, cancerof larger cells of the lungs, cancer of the colon, cancer of the centralnervous system, melanoma, cancer of the ovaries, cancer of the kidney,cancer of the prostate and cancer of the breast). All the cell lines hadan equivalent sensitivity to roscovitine. The mean IC₅₀ value is 16 μM(whereas it is 60.3 μM for olomoucine). No correlation was found betweenthe sensitivity of the cell lines to roscovitine and the presence ofwild or muted p53. The method of comparison analysis showed that theeffects of roscovitine and of flavopiridol are comparable.

As regards the effects of roscovitine on the growth of the cell lineL1210, a very clear dose-dependent inhibition of the growth was found,as shown on FIG. 7A, where the cell growth is shown as a function of theconcentration of roscovitine or olomoucine. The curves are largelyidentical after two and three days of culture, as found with theabovementioned tumoral cells. Roscovitine is approximately four timesmore effective than olomoucine in inhibiting cell growth (IC₅₀ of 40 μMfor roscovitine and 160 μM for olomoucine). Although the majority ofcells are viable (96±2% by Trypan blue exclusion) after a treatment with60 μM roscovitine for 48 hours, they remain irreversibly stopped, evenafter extensive washings. The cells exposed to 120 μM roscovitine dierapidly. The effects of roscovitine on the distribution of the cellcycle were then studied by flow cytometry. At 60 μM roscovitine, thecells remain stopped at G1 and accumulate in G2 as shown in FIG. 7B,where the proportions (%) of each phase of the cell cycle observed (G1,S, G2/M) in the presence or absence of roscovitine are shown.

4A4 antibodies were used with the aim of identifying the moleculartarget of roscovitine in vivo. The results are illustrated in FIG. 8,where the total proteins extracted from cells treated (÷) or not treated(−) with roscovitine and then resolved on SDS-PAGE before westerntransfer with the 4A4 antibodies are shown. The non-treated cells stopin the metaphase and accumulate vimentine phosphorylated by cdc2. Thecells treated with roscovitine on the other hand, do not have vimentinephosphorylated by cdc2, which shows that cdc2 has in fact been inhibitedin vivo and that the cells were stopped before the metaphase.

Roscovitine also helps to reduce the hyperphosphorylation of tauobserved during Alzheimer's disease: a cdk specific to thebrain(cdk5/p35 ) which phosphorylates certain sites of tau isparticularly sensitive to roscovitine.

BIBLIOGRAPHIC REFERENCES

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What is claimed is: 1.2-(1-D,L-Hydroxymethylpropylamino)-6-benzylamino-9-isopropylpurine. 2.6-benzylamino-2-R-(1-ethyl-2-hydroxyethylamino)-9-isopropyl purine.
 3. Apharmaceutical composition comprising a therapeutically effective amountof the compound of claim 1 and a pharmaceutical vehicle.
 4. Apharmaceutical composition comprising a therapeutically effective amountof the compound of claim 2 and a pharmaceutical vehicle.